JP6960399B2 - How to treat or prevent dry eye - Google Patents

Description

The present invention relates to a method for treating and preventing dry eye with a compound which is 2,3,5,4'-tetrahydroxystilbene 2-O-β-D-glucopyranoside.

Xerophthalmia is commonly known as keratoconjunctivitis sicca. Millions of people suffer from this common eye disease each year. There are many factors that can affect the risk of xerophthalmia. The rapid development of electronic products is the most common reason for xerophthalmia today. The number of patients with xerophthalmia has increased significantly due to the increase in smartphones, tablets and laptops. Therefore, the development of treatment and prevention methods for xerophthalmia is an important research theme.

According to the American Ophthalmology Society, xerophthalmia is either (1) decreased tear production or (2) excessive tear evaporation in the normal tear film, that is, the mucus commonly found in the tear film. It is defined as a disease with abnormal lipid production. In addition, xerophthalmia can be classified into different levels based on symptoms and severity. Dry eye includes symptoms such as dry eyes, itching, inflammation, drowsy eyes, excess tears, tingling and tingling pain, foreign body sensations, dazzling light, and blurred vision. When the symptoms become severe, the eyes may swell, become congested, and have impaired vision.

Specific risk factors for dry eye include aging, undersecretion of male hormones in postmenopausal women, undernutrition, alcohol dependence, corneal damage caused by infection or chemical burns, and eyeballs with hyperthyroidism. Includes protrusion, facial or trigeminal spasm, long-term contact lens wear, and autoimmune disorders such as Sjogren's syndrome, systemic lupus erythematosus, erythema polymorphism, and rheumatoid arthritis.

Standard treatments for xerophthalmia include artificial tears, surgical removal of the punctal plug, and corticosteroid eye drops. In addition, in 2003, 0.05% cyclosporine was approved by the FDA for the treatment of inflammation and androgen deficiency-induced xerophthalmia. While these treatments can keep the eyes moist, prevent tear evaporation, and relieve dryness of the eye surface, frequent use of these agents only increases the risk of toxic effects and irritation. It may lead to an increase in drug-resistant infections. As a result, non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly being used to treat dry eye instead of steroids.

Nerve growth factor (NGF), discovered in the 1950s, is important for the growth, maintenance, and survival of specific target nerves (nerve cells). They also function as signal transduction substances. NGF has two receptors, Trk A and p75 NTR. NGF and its receptors have also been demonstrated to be expressed by rat lacrimal gland tissue and have been quantified in human tear fluid (Lee, HK, et al., Am J Ophthalmol, 2005). .139 (6): p.965-71). In addition, NGF has been shown to induce proliferation and differentiation of corneal epithelial cells in vitro and in the tear film and corneal epithelium, which play important roles in eye surface maintenance and corneal wound healing (Lambise, A., et al., Invest Opthelmol Vis Sci, 2009.50 (10): p.4622-30). NGF eye drops have been shown to be able to increase tear secretion and conjunctival goblet cell density in canine dry eye models (Coassin, M., et al., Graefes Arch Clin Exp Ophthalmol, 2005.243 (2): p. .151-5). In clinical practice, patients with dry eye have increased expression of NGF in tears, which is a compensatory mechanism for patients with dry eye to maintain normal functioning of the ocular surface (Lambiase, A., et al. , Arch Ophthalmol, 2011.129 (8): p.981-6). In addition, NGF can induce the differentiation of conjunctival epithelial cells into goblet cells capable of secreting mucin to protect the ocular surface (Lambise, A., et al., Invest Opphalmol Vis Sci, 2009.50 (10). ): P.4622-30; Lambise, A., et al., Proc Natl Acad Sci USA, 2009.106 (32): p.13469-74). However, these results suggest that regulation of NGF is an important factor in the treatment of dry eye and eye protection.

There is still a need for safer and more effective treatments for dry eye.

Lee, H. K. , Et al. , Am J Ophthalmol, 2005.139 (6): p. 965-71 Lambise, A. , Et al. , Invest Opthalmol Vis Sci, 2009.50 (10): p. 4622-30 Coassin, M.D. , Et al. , Graefes Arch Clin Exp Ophthalmol, 2005.243 (2): p.151-5 Lambise, A. , Et al. , Arch Ophthalmol, 2011.129 (8): p. 981-6 Lambise, A. , Et al. , Invest Opthalmol Vis Sci, 2009.50 (10): p. 4622-30; Lambise, A. , Et al. , Proc Natl Acad Sci USA, 2009.106 (32): p. 13469-74

An object of the present invention is to provide a method for treating and preventing dry eye with a compound which is 2,3,5,4'-tetrahydroxystilbene 2-O-β-D-glucopyranoside.

式（Ｉ）、
ここで、Ｒはグルコースである。 The method for treating or preventing dry eye of the present invention comprises the step of administering to a subject in need an effective amount of a compound represented by the general formula (I).

Equation (I),
Here, R is glucose.

In certain embodiments of the invention, the compound of formula (I) is administered in an amount effective to increase tear secretion in the subject.

In certain embodiments of the invention, the compound of formula (I) is administered in an amount effective to increase the endogenous NGF secretion of the subject's lacrimal tissue.

It should be understood that the above description of the outline and the following detailed description are not intended to limit the present invention and are for the purpose of illustration and description only.

The above-mentioned outline of the present invention and the following detailed description will be better understood by reading with reference to the accompanying drawings. For purposes of exemplifying the present invention, currently preferred embodiments are described in the drawings.

FIG. 5 is a graph showing the effect of the compound of formula (I) on BAC-induced injury in HCE cells. 6 is a graph showing the improvement of tear secretion of the compound of formula (I) in the BAC-induced dry eye mouse model (1). 6 is a graph showing the improvement of tear secretion of the compound of formula (I) in the BAC-induced dry eye mouse model (2). FIG. 5 is a graph showing the effect of the compound of formula (I) on tear NGF protein expression in BAC-induced dry eye mice.

The terms used herein have general meaning in the art in general within the context of the present invention and in the particular context in which each term is used. Specific terms used to describe the invention are described below or elsewhere herein to provide the practitioner with additional guidance regarding the description of the invention. The use of examples in all parts of this specification is for illustration purposes only, including examples of all terms described herein, and in any way limits the gist and meaning of the present invention, or any of the exemplified terms. It's not something to do. Similarly, the invention is not limited to the various embodiments presented herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of inconsistency, this document shall be followed, including definitions.

As used herein, "roughly", "about", or "approximately" generally means within 20 percent, preferably within 10 percent, and more preferably within 5 percent of the values or ranges indicated. .. The quantities shown herein are approximate, that is, they may be implied even if the terms "roughly", "about", or "approximately" are not explicitly stated.

式（Ｉ）、
ここで、Ｒはグルコースである。 In one aspect, the invention provides a method of treating or preventing dry eye. The method of the present invention comprises the step of administering to a required subject an effective amount of a compound represented by the general formula (I).

Equation (I),
Here, R is glucose.

The compounds of formula (I) can be made by methods known in the art, for example, methods disclosed in US Pat. No. 2014160243 A1, the entire patent being incorporated herein by reference.

As used herein, the term "effective amount" refers to an amount sufficient to provide the desired therapeutic or preventive effect of a compound of formula (I), or the induction of a particular type of response. The required effective amount will vary from subject to subject, depending on the subject's disease status, physical condition, age, gender, race, weight, and the like. However, a suitable effective amount could be determined by one of ordinary skill in the art using only conventional experiments. In certain embodiments of the invention, the effective amount is an amount effective for increasing tear secretion in a subject. In certain embodiments of the invention, said effective amount is an amount effective for increasing the endogenous NGF secretion of a subject's lacrimal tissue (or lacrimal gland).

The compound of formula (I) can be instilled into a subject 1 to 10 times a day in an amount of 1 to 10 drops at a time, preferably 1 to 4 times a day in an amount of 1 to 5 drops at a time. For example, the subject can use 3 drops of the preparation containing the compound of the formula (I) each time and 3 times a day. For topical administration to the eye, a compound of formula (I) having a concentration of 1 to 20 μg / ml can be used, and preferably a compound of formula (I) having a concentration of 2 to 10 μg / ml can be used. ..

Preferably, the compound of formula (I) is administered topically to the subject's eye.

In another aspect, the invention provides a topical ophthalmic composition for treating or preventing dry eye, comprising a compound of formula (I) and a pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared by conventionally known methods using one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier. Such carriers can include, but are limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, propylene glycol, cremofol, nanoparticles, liposomes, polymers, and combinations thereof. No.

In yet another aspect, the invention provides the use of compounds of formula (I) in the manufacture of agents for the treatment or prevention of dry eye.

Hereinafter, the present invention will be further described with reference to the following examples provided for purposes of illustration, but not limitation.

Example Benzalkonium chloride (BAC) is one of the most commonly used preservatives in eye drops. However, this preservative is recognized as a potential risk of xerophthalmia. Therefore, many in vitro and in vivo models of xerophthalmia have been successfully developed by BAC.

Based on these models, there is strong experimental evidence to prove that the compounds of formula (I) are effective eye drops for xerophthalmia. Human corneal epithelial cells were first injured with benzalkonium chloride and then treated with a compound of formula (I), and it was discovered that the compound of formula (I) enhances cell viability after injury. In addition, benzalkonium chloride was used as a dry eye inducer in the mouse model. After a week of injury, mice were treated with eye drops containing 2, 5 or 10 μg / ml of compound of formula (I) three times daily, which restored tear secretion and increased NGF gene expression levels. I found that I did. The results showed that the compound of formula (I) could activate NGF in the lacrimal gland, increase tear secretion and repair damage to the ocular surface.

Effect of compound of formula (I) on BAC-induced injury in HCE cells

Benzalkonium chloride cell damage model

Human corneal epithelial cells (HCE) ⁵ × 10 5 cells in cell 6-well plates were seeded 24 hours at 10% FCS-containing DMEM / F12 medium. After cell adhesion, the medium is removed, transferred to 0.0005% benzalkonium chloride, 24 hours later, washed with PBS, compounds of formula (I) at different concentrations of 0.4, 2, 10 μg / ml ( It was treated with compound A) for 48 hours. Finally, the cells were trypsinized and centrifuged at 1000 rpm for 5 minutes, then the supernatant was removed and 1 ml DMEM / F12 medium was added to resuspend the cells. Subsequently, 0.4% trypan blue and the medium were diluted 1: 3, and the viability of treatment with different concentrations was observed by counting the number of cells under a microscope. The results are shown in FIG. * P <0.05, ** P <0.01, compared with group A of 0 g / ml compound, Student's T-test. The data are shown as mean ± SD (n = 3).

Compound of formula (I) improves tear secretion in BAC-induced dry eye mice

Benzalkonium chloride-induced dry eye mouse model (1)

Male BALB / c mice (6 weeks of age) were treated with local administration of 5 μl of 0.3% BAC to the right eye three times daily (9:00, 13:30, 18:00). Mice were treated with PBS as a control group from day 1 to day 7. Then, 5 μl of compound A (2, 5, 10 μg / ml) was locally administered to the right eye three times a day (9:00, 13:30, 18:00), and the control group was administered from the 8th day to the 19th day. Treated with PBS.

The phenol red cotton thread tear test method was used. The cotton thread is yellow (acidic) and turns pale red when in contact with tears. Using forceps, the bent portion of the cotton thread was inserted into the conjunctiva of the eyelid of the eye at a distance of 1/3 from the outer eye angle of the lower eyelid. After 15 seconds, the cotton thread was removed and the entire wet area (red) was measured. Phenol red cotton thread tear tests were performed on days 7, 11, 15, and 19. The results are shown in FIG. * P <0.05, ** P <0.01, compared with control group, Student's T-test. The data are shown with mean ± SD (n = 5-6).

Benzalkonium chloride-induced dry eye mouse model (2)

Male BALB / c mice (9 weeks of age) were treated with local administration of 5 μl of 0.3% BAC to the right eye three times daily (9:00, 13:30, 18:00). Mice were treated with PBS as a control group from day 1 to day 7. Then, 5 μl of compound A (5, 10 μg / ml) was locally administered to the right eye three times a day (9:00, 13:30, 18:00), and the control group was treated with PBS from the 15th day to the 19th day. I was treated.

The phenol red cotton thread tear test method was used. The cotton thread is yellow (acidic) and turns pale red when in contact with tears. Using forceps, the bent portion of the cotton thread was inserted into the conjunctiva of the eyelid of the eye at a distance of 1/3 from the outer eye angle of the lower eyelid. After 15 seconds, the cotton thread was removed and the entire wet area (red) was measured. Phenol red cotton thread tear tests were performed on days 14, 18 and 20. The results are shown in FIG. * P <0.05, ** P <0.01, compared with control group, Student's T-test. The data are shown with mean ± SD (n = 5-6).

Effect of Compound of Formula (I) on Tear NGF Protein Expression in BAC-Induced Dry Eye Mice

Tear fluid was collected from the right eye of BAC-induced dry eye mice by washing 10 μl of PBS. Mice were treated with compound A eye drops at a different concentration than PBS (control group) for 10 days. The NGF protein was a positive control. A tear volume of 5 μl was used in the Western blot.

Total protein concentration was measured in the 1x Braford assay. For denaturation, a loading buffer (7 ml 0.5 M Tris pH 6.8, 3 ml glycerol, 1 g 10% SDS, 0.93 g DTT, 0.5 mg 0.05% bromophenol blue, 10 ml) was used. , The mixture was boiled at 95-100 ° C. for 5 minutes. For electrophoresis, 20-40 μg of total protein was loaded per minigel (12% SDS polyacrylamide gel) well. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane, which was blocked in TTBS with 5-10% nonfat milk powder. Membranes were incubated with the primary antibody by shaking overnight at 4 ° C., washed multiple times with TTBS to remove residual primary antibody, and then incubated with the secondary antibody for 2 hours at room temperature.

Protein levels were measured by a chemiluminescent detection system and analyzed with Imagequant software. The results are shown in FIG. * P <0.05, ** P <0.01, *** P <0.001, compared with control group, Student's T-test. The data are shown as mean ± SD (n = 4).

It will be apparent to those skilled in the art that modifications can be made to the embodiments described above without departing from the broader concepts of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed, and it is understood that the gist of the present invention and modifications within the scope of the invention as defined by the appended claims are included in the present invention.

Claims (4)

In the manufacture of a medicament for increasing tear secretion or endogenous NGF secretion in a subject's eye, formula (I):

Equation (I)
Use of 2,3,5,4'-tetrahydroxystilbene 2-O-β-D-glucopyranoside , a compound represented by. The use according to claim 1, wherein the medicament is for topical administration to the eyes of a subject. The use according to claim 2 , wherein the subject is a subject suffering from dry eye. A topical ophthalmic composition for increasing tear secretion or endogenous NGF secretion in the eye, of formula (I) :.

In a compound represented by 2,3,5,4'- and tetrahydroxy stilbene 2-O-β-D- glucopyranoside, a pharmaceutically acceptable carrier including, eye tear secretion or endogenous A topical ophthalmic composition for increasing NGF secretion.

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